Introduction:
Column chromatography is a widely used separation technique in which the mobile phase carrying the sample is allowed to pass through a stationary phase contained within a column.
The method exploits differences in compound affinity towards the stationary phase. In adsorption column chromatography, the stationary phase is a solid, and compounds get separated based on their adsorption on the solid surface
Methodology:
Adsorption column chromatography is a technique that exploits the differential affinities of compounds towards a solid stationary phase.
Here's a step-by-step breakdown of the process:
1. Preparation of the Column:
A glass column is packed with a stationary phase, typically an adsorbent like silica gel (SiO2) or alumina (Al2O3).
A piece of cotton or a small plug is placed at the bottom to prevent the stationary phase from flowing out.
The adsorbent is then slurred in a suitable solvent (eluent) and poured into the column, ensuring that no air bubbles are trapped.
The column is then topped with a layer of sand to prevent disturbance of the stationary phase when adding samples or solvents.
2. Sample Loading:
The mixture to be separated is loaded onto the top of the column.
This can be done by dissolving the mixture in a minimal amount of solvent and carefully adding it to the column or by applying a dried sample onto the column if pre-adsorbed on a small amount of stationary phase.
3. Elution:
The mobile phase (solvent) is poured onto the column. As it travels down, it carries the sample with it.
Components in the mixture will have different affinities for the stationary phase.
Those with lower affinity will travel faster and elute first, while those with higher affinity will be retained longer and elute later.
4. Detection & Collection:
Fractions are collected at the bottom of the column in test tubes or vials. Depending on the setup, one may rely on visual indicators (like colored bands) or use instruments like UV-Vis detectors to detect the eluting compounds.
5. Analysis:
The collected fractions are then analyzed to determine which components they contain.
Thin-layer chromatography (TLC) is commonly used for this purpose.
Components:
Stationary Phase: Commonly used stationary phases are silica gel (SiO2) or alumina (Al2O3). These materials have surface hydroxyl groups that can form weak interactions (van der Waals forces, hydrogen bonding, etc.) with the sample molecules.
Mobile Phase: Organic solvents, such as hexane, ether, or chloroform, are typically used. The polarity and composition of the mobile phase can be adjusted to optimize separation.